Reaction mass of Phosphorous(1+), (N-ethylethanaminato) diphenyl(phenylmethyl)-(T-4)-salt with 4, 4' - [2,2,2-trifluoro-1-(trifluoromethyl) ethylidene bis[phenol] (1:1) and (4,4'-[2,2,2-Trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2013 - April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- /batch No.of test material: 3706OB
- Expiration date of the batch: 31 December 2018
- Purity test date :Not applicable -complex mixture (> 99%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range finding test:
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive test:
Samples were taken from the control (replicates R1 - R6 pooled) and each loading rate WAF test group (replicates Ri - R3 pooled) at 0 and 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting/shielded from the light

Range finding test:
-First range finding test:
Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions or particles of test item present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

-Second range finding test:
An amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions or particles of test item present. A series of dilutions was made from the 1.0 mg/L loading rate WAF after filtering to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.8 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF

Defintive test:
Due to the need to test at relatively low test concentrations, WAFs were prepared at nominal loading rates of 1.0, 3.2 and 10 mg/L. A dilution was made from the 1.0 and 3.2 mg/L loading rate WAFs to give the 0.10 and 0.32 mg/L loading rate WAF test preparations respectively.
Amounts of test item (20, 64 and 200 mg) were each separately added to the surface of 20, 20 and 10 liters of culture medium to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions or particles of test item present. A dilution was made from the 1.0 and 3.2 mg/L loading rate WAFs to give further test concentrations of 0.10 and 0.32 mg/L loading rate WAF respectively.
An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata strain
- Strain: CCAP 278/4
- Source : Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10xE4 — 10xE5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no data reported

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH ranged from 7.6 to 8.6 during the definitive test

Dissolved oxygen:

no data reported

Salinity:

no data reported

Conductivity:

no data reported

Nominal and measured concentrations:

Range finding test:
-first range finding test: 0, 10 and 100 mg/L loading rate WAF
-Second range finding test: of 0.010, 0.10 and 1.0 mg/L loading rate WAF

Definitive test:
0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type : closed, plugged with polyurethane foam bungs
- Fill volume: 100 mL of test preparation
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.
- Renewal rate of test solution: no
- Initial cells density: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10XE3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10xE4 — 10XE5 cells/mL.
- No. of vessels per concentration (replicates):
>range finding tests : 2 replicates per test group
>Defintive test: 3 replicates
- No. of vessels per control (replicates):
> range finding tests: 2 replicates
> Defintive test: 6 replicates
- exposure conditions
> range finding tests: The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
> Defintive test:
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 8.25 x 10xE5 cells per mL. Inoculation of 1 liter of test medium with 6.1 mL of this algal suspension gave an initial nominal cell density of 5 x 10xE3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.


GROWTH MEDIUM
- Standard medium used: yes , The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:continuous illumination
- Light intensity and quality: (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations:
> Range finding test: At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
> Defintive test: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST MEDIUM / WATER PARAMETERS
The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.



TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: -first range finding test: 0, 10 and 100 mg/L loading rate WAF/ -Second range finding test: of 0.010, 0.10 and 1.0 mg/L loading rate WAF
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF Water Accommodated Fraction

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: WAF Water Accommodated Fraction

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF Water Accommodated Fraction

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: WAF Water Accommodated Fraction

Details on results:

RANGE FINDING TEST:
The results showed no effect on growth at 0.010 and 0.10 mg/L loading rate WAF. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF. See table 1 and 2 in 'any other information on results incl. tables"
Chemical analysis of the test preparations taken from the second range-finding test at 0 and 72 hours (see Appendix 4) showed measured test concentrations to range from 0.00092 to 0.0052 mg/L and from 0.0013 to 0.0085 mg/L respectively.

DEFINITIVE TEST: See table 3 and 4 in 'any other information on results incl. tables"
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
- Inhibition of growth rate:
ErL10 (0 - 72 h) : 1.8 mg/L loading rate WAF
ErL20 (0 - 72 h) : 2.6 mg/L loading rate WAF
ErL50 (0 - 72 h) : 5.2 mg/L loading rate WAF (It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.)
There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P>0.05), however all other loading rates were significantly different (PO.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 3.2 mg/L loading rate WAF.

- Inhibition of Yield:
EyL10 (0 - 72 h) : 0.95 mg/L loading rate WAF
EyL20 (0 - 72 h) : 1.3 mg/L loading rate WAF
EyL50(0-72h) : 2.1 mg/L loading rate WAF; 95% confidence limits 1.8 - 2.5 mg/L loading rate WAF

There were no statistically significant decreases in yield between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P>0.05), however all other loading rates were significantly different (PO.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 3.2 mg/L loading rate WAF.

-Chemical Analysis of Test Loading Rates: Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.00027 mg/L, at 0.10 mg/L loading rate WAF through to 0.0090 mg/L at 10 mg/L loading rate WAF. Concentrations in the range of 0.00029 to 0.010 mg/L were observed at 72 hours.
The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

OBSERVATION ON CULTURES:
After 72 hours there were no abnormalities detected in the control or test cultures at 0.10, 0.32, 1.0 and 3.2 mg/L loading rate WAF, however cell debris and swollen cells were observed to be present in the test cultures at 10 mg/L loading rate WAF.

OBSERVATION ON TEST ITEM SOLUBILITY:
Observations on the test media were carried out during the mixing and testing of the WAFs.At the start of mixing, all loading rate WAFs were observed to be clear colorless media columns with particles of test item floating at the media surface. After stirring, all loading rate WAFs were observed to have formed clear colorless media columns with particles of test item floating at the surface and dispersed throughout. After standing for 1-Hour all loading rate WAFs were observed to have formed clear colorless media columns with particles of test item floating at the surface, dispersed throughout the media column and settled on the bottom of the mixing vessel.
As visual observations made on the WAFs indicated that a significant amount of dispersed test item was present, it was considered appropriate to filter the WAFs through a glass wool plug. Microscopic examination of the WAFs after filtering showed that there were no micro-dispersions of particles of test item present.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period, all control, 0.10 and 0.32 mg/L loading rate WAF test cultures were observed to be green dispersions. The 1.0 mg/L loading rate WAF test cultures were observed to be pale green dispersions, the 3.2 mg/L loading rate WAF test cultures were very pale green dispersions whilst the 10 mg/L loading rate WAF test cultures were clear colorless solutions.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41300098) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 - 72 h) : 1.2 mg/L; 95% confidence limits 1.0-1.3 mg/L
EyC50 (0 - 72 h) : 0.52 mg/L; 95% confidence limits 0.45 - 0.62 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data were carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Table 1     Cell Densities and Percentage Inhibition of Growth from the Initial Range-finding Test

Nominal Loading Rate (mg/L)		Cell Densities*	(cells per mL)	Inhibition Values (%)	Cell Densities*
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.34E+03	2.31E+04**	-	-
	R2	5.92E+03	1.09E+06
	Mean	6.13E+03	5.55E+05
10	Ri	5.87E+03	2.63E+04	67	96
	R2	5.44E+03	2.63E+04
	Mean	5.66E+03	2.63E+04
100	R1	4.22E+03	3.57E+03	110	100
	R2	5.71E+03	3.06E+03
	Mean	4.97E+03	3.31E+03

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

**Replicate considered to be contaminated due to significantly lower measured cell density compared to replicate R2

Table 2     Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Loading Rate (mg/L		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	Ri	5.17E+03	6.88E+05	-	-
	R2	5.71E+03	6.72E+05
	Mean	5.44E+03	6.80E+05
0.010	Ri	5.28E+03	5.88E+05	1	4
	R2	6.00E+03	7.17E+05
	Mean	5.64E+03	6.52E+05
0.10	Ri	4.98E+03	7.05E+05	[3]	[8]
	R2	5.54E+03	7.58E+05
	Mean	5.26E+03	7.31E+05
1.0	R1	5.52E+03	9.22E+04	43	88
	R2	5.26E+03	7.44E+04
	Mean	5.39E+03	8.33E+04

Table 3:    Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate		pH	Cell Densities* (cells per mL)				pH
(mg/L)		0h	0h	24h	48h	72h	72 h
Control	R1 R2 R3 R4 R5 R6 Mean	7.8	4.18E+03 5.14E+03 4.70E+03 4.53E+03 4.58E+03 5.57E+03 4.78E+03	2.88E+04 2.95E+04 2.81E+04 2.59E+04 2.97E+04 2.93E+04 2.86E+04	1.62E+05 1.73E+05 1.62E+05 1.53E+05 1.57E+05 1.69E+05 1.63E+05	8.20E+05 8.42E+05 7.31E+05 7.38E+05 6.55E+05 7.83E+05 7.62E+05	8.4
0.10	R1 R2 R3 Mean	7.8	6.69E+03 5.94E+03 6.05E+03 6.22E+03	3.12E+04 3.50E+04 3.06E+04 3.22E+04	1.89E+05 1.91E+05 1.92E+05 1.91E+05	8.46E+05 9.19E+05 8.46E+05 8.70E+05	8.4
0.32	R1 R2 R3 Mean	7.8	6.62E+03 6.42E+03 6.19E+03 6.41E+03	2.99E+04 2.73E+04 3.06E+04 2.93E+04	1.71E+05 1.46E+05 1.83E+05 1.67E+05	7.44E+05 6.73E+05 7.92E+05 7.37E+05	8.1
1.0	R1 R2 R3 Mean	7.7	5.20E+03 5.09E+03 4.59E+03 4.96E+03	2.81E+04 2.89E+04 2.98E+04 2.89E+04	1.48E+05 1.49E+05 1.69E+05 1.55E+05	6.86E+05 6.74E+05 6.68E+05 6.76E+05	8.6
3.2	R1 R2 R3 Mean	7.6	4.43E+03 5.31E+03 5.50E+03 5.08E+03	2.32E+04 1.97E+04 2.42E+04 2.24E+04	7.98E+04 7.11E+04 7.17E+04 7.42E+04	2.26E+05 1.79E+05 1.82E+05 1.95E+05	8.0
10	R1 R2 R3 Mean	7.6	5.49E+03 5.42E+03 5.38E+03 5.43E+03	1.10E+04 8.62E+03 9.60E+03 9.75E+03	9.73E+03 8.24E+03 6.88E+03 8.28E+03	1.68E+04 1.50E+04 1.28E+04 1.49E+04	8.0

Table 4:      Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate                 (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0  72 h	% Inhibition	0-72h	% Inhibition*
Control	R1	0.071		8.16E+05
	R2	0.071		8.37E+05
	R3	0.069		7.26E+05
	R4	0.069	-	7.33E+05	|
	R5	0.068		6.51E+05
	R6	0.070		7.78E+05
	Mean	0.070		7.57E+05
	SD	0.001		6.79E+04
0.10	R1	0.071	[1]	8.39E+05
	R2	0.072	[3]	9.13E+05
	R3	0.071	[1]	8.40E+05
	Mean	0.071		8.64E+05	[14]
	SD	0.001		4.25E+04
0.32	R1	0.069	1	7.38E+05
	R2	0.068	3	6.67E+05
	R3	0.070	0	7.86E+05
	Mean	0.069	1	7.30E+05	4
	SD	0.001		5.98E+04
1.0	R1	0.068	3	6.81E+05
	R2	0.068	3	6.69E+05
	R3	0.068	3	6.64E+05
	Mean	0.068	3	6.71E+05	11
	SD	0.000		9.06E+03
3.2	R1	0.053	24	2.21E+05
	R2	0.050	29	1.73E+05
	R3	0.050	29	1.76E+05
	Mean	0.051	27	1.90E+05	75
	SD	0.002		2.68E+04
10	R1	0.017	76	1.13E+04
	R2	0.015	79	9.60E+03
	R3	0.013	81	7.41E+03
	Mean	0.015	79	9.42E+03	99
	SD	0.002		1.93E+03

[Increase in growth as compared to controls]

Analytical Results:

Table 5:     Results for Test Samples (Range-Finding test)

Time point      [hours]	NominalLoading Rate Cnom [mg/L]	Measured Concentration of Test Item in Sample Vial X [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item in Range-Finding Sample C [mg/L]
0	0.10 1.0 10	0.0366 0.194 0.208	0.025 0.025 0.025	0.000916 0.00484 0.00521
72	0.10 1.0 10	0.0536 0.226 0.338	0.025 0.025 0.025	0.00134 0.00566 0.00846

Table 6:    Results for Test Samples (definitive test)

Time point [hours]	NominalLoading Rate Cnom [mg/L]	Measured Concentration of Test Item in Sample Vial X [mg/L]	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample C  [mg/L]
0	Control 0.10 0.32 1.0 3.2 10	<LOQ <LOQ/0.0234** */<LOQ** 0.0259 0.0972 0.359	0.025 0.025 0.025 0.025 0.025 0.025	<LOQ <LOQ/0.000586** */<LOQ** 0.000648 0.00243 0.00897
72	Control 0.10 0.32 1.0 3.2 10	<LOQ 0.0255 0.0116 0.0336 0.109 0.404	0.025 0.025 0.025 0.025 0.025 0.025	<LOQ 0.000637 0.000291 0.000841 0.00272 0.0101

* =  Atypical response seen on chromatogram, no result reported

** =  Duplicate samples, stored frozen prior to analysis

LOQ = Limit of Quantification

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
The ErL50 (72h) was 5.2 mg/L for the growth rate.
The EyL50 (72h) was 2.1 mg/L for the yield.
The NOELR (72) was 1.0 mg/L for both growth rate and yield.
The LOELR (72) was 3.2 mg/L for both growth rate and yield.

Executive summary:

The effect of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to OECD Guideline 201 (2006), and the method C.3. of Commission Regulation (EC) No 440/2008, C.3. The study was compliant with the GLP.

Due to the low aqueous solubility and complex nature of the test item, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), thus all the test solutions were prepared as a Water Accommodated Fraction (WAF). 

 

Based on results of a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24± 1 °C.

Samples of the algal populations were removed daily and Algal biomass determined for each control and treatment group, using a Coulter® Multisizer Particle Counter

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.00027 mg/L) at 0.10 mg/L loading rate WAF through to 0.0090 mg/L at 10 mg/L loading rate WAF. Concentrations in the range of 0.00029 to 0.010 mg/L were observed at 72 hours. The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

 

The results obtained with loading rates were as follows:

72h-ErL50 = 5.2 mg/L (growth rate)

72h-EyL50 = 2.1 mg/L (yield)

72h-NOELR = 1.0 mg/L (growth rate and yield)

72-hLOELR = 3.2 mg/L (growth rate and yield)

 

In addition, the microscopic examination (shape and size) of the algal cells at the end of the test showed no abnormalities in the control or test cultures at 0.10, 0.32, 1.0 and 3.2 mg/L loading rate WAF, however cell debris and swollen cells were observed to be present in the test cultures at 10 mg/L loading rate WAF.

 

The three validity criteria of the OECD guideline 201 were fulfilled, therefore this study is considered as reliable without restrictions.

 

Description of key information

One reliable study is available for the Reaction mass of AminoPhosphonium salt and BisphenolAF (Xa 31) for this endpoint. The effect of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to OECD Guideline 201 (2006), and the method C.3. of Commission Regulation (EC) No 440/2008, C.3. The study was compliant with the GLP. Due to the low aqueous solubility and complex nature of the test item, the test was performed using the loading rate (according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000), thus all the test solutions were prepared as a Water Accommodated Fraction (WAF). 

 

Based on results of a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24± 1 °C. Samples of the algal populations were removed daily and Algal biomass determined for each control and treatment group, usinga Coulter® Multisizer Particle Counter

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.00027 mg/L) at 0.10 mg/L loading rate WAF through to 0.0090 mg/L at 10 mg/L loading rate WAF. Concentrations in the range of 0.00029 to 0.010 mg/L were observed at 72 hours. The toxicity of the test item cannot be attributed to a single component or a mixture of components but to the test item as a whole. In addition, the measured concentrations obtained for the WAFs are not stability indicating and are rather a confirmation that the test item is present. Given these factors the results of the study were based on nominal loading rates only.

 

The results obtained with loading rateswere as follows:

72h-ErL50 = 5.2 mg/L (growth rate)

72h-EyL50 = 2.1 mg/L (yield)

72h-NOELR = 1.0 mg/L (growth rate and yield)

72-hLOELR = 3.2 mg/L (growth rate and yield)

 

In addition, the microscopic examination (shape and size) of the algal cells at the end of the test showed no abnormalities in the control or test cultures at 0.10, 0.32, 1.0 and 3.2 mg/L loading rate WAF, however cell debris and swollen cells were observed to be present in the test cultures at 10 mg/L loading rate WAF.

Update (2018). As the algal ECx values are based on nominal loading rates, they may lead to a possible misunderestimation of toxic effects, especially for chronic ones. One of the main constituent of the reaction mass is bisphenol AF (CAS RN 1478-61-1) which has been registered under the REACH Regulation in 2018. According to the data reported, bisphenol AF exhibits an aquatic toxicity profile similar to the one of the reaction mass (i.e. EC/LC50 in the range >1-10 mg/L for fish, daphnids and algae) except for the chronic NOEC value on algae. Whereas a 72h-NOELR of 1 mg/L is reported for the reaction mass (growth rate, based on loading rates), a 72h-NOEC value of 0.052 mg/L is reported for bisphenol AF (growth rate, based on analytically confirmed concentrations). Considering the analytical deficiencies of the algal toxicity study performed on the reaction mass, it is found preferable to use for the assessment, as a worst-case, the 72h-NOEC value reported for bisphenol AF. This worst-case approach is all-the-more relevant as the aquatic (chronic) toxicity of the reaction mass is not expected to be driven by the other consituent of the reaction mass: the aminophosphonium salt (ECx values ≥ 1 mg/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

5.2 mg/L